Bowtie 2 seems to be working fine (tested command '/home/shared/bowtie2-2.4.4-linux-x86_64/bowtie2 --version' [2.4.4]) Output format is BAM (default) Alignments will be written out in BAM format. Samtools found here: '/usr/bin/samtools' Reference genome folder provided is ../data/ (absolute path is '/home/shared/16TB_HDD_01/sr320/github/project-mytilus-methylation/data/)' FastQ format assumed (by default) Attention: using more than 4 cores per alignment thread has been reported to have diminishing returns. If possible try to limit -p to a value of 4 Each Bowtie 2 instance is going to be run with 8 threads. Please monitor performance closely and tune down if necessary! Input files to be analysed (in current folder '/home/shared/16TB_HDD_01/sr320/github/project-mytilus-methylation/code'): ../data/95M_R1.fastp-trim.fq.gz ../data/95M_R2.fastp-trim.fq.gz Library is assumed to be strand-specific (directional), alignments to strands complementary to the original top or bottom strands will be ignored (i.e. not performed!) Output will be written into the directory: /home/shared/16TB_HDD_01/sr320/github/project-mytilus-methylation/output/01.2-bismark/ Setting parallelization to single-threaded (default) Summary of all aligner options: -q --score-min L,0,-1.0 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 Current working directory is: /home/shared/16TB_HDD_01/sr320/github/project-mytilus-methylation/code Now reading in and storing sequence information of the genome specified in: /home/shared/16TB_HDD_01/sr320/github/project-mytilus-methylation/data/ Single-core mode: setting pid to 1 Paired-end alignments will be performed ======================================= The provided filenames for paired-end alignments are ../data/95M_R1.fastp-trim.fq.gz and ../data/95M_R2.fastp-trim.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file 95M_R1.fastp-trim.fq.gz to 95M_R1.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 95M_R1.fastp-trim.fq.gz (85285639 sequences in total) Writing a G -> A converted version of the input file 95M_R2.fastp-trim.fq.gz to 95M_R2.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 95M_R2.fastp-trim.fq.gz (85285639 sequences in total) Input files are 95M_R1.fastp-trim.fq.gz_C_to_T.fastq and 95M_R2.fastp-trim.fq.gz_G_to_A.fastq (FastQ) Now running 2 instances of Bowtie 2 against the bisulfite genome of /home/shared/16TB_HDD_01/sr320/github/project-mytilus-methylation/data/ with the specified options: -q --score-min L,0,-1.0 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 Now starting a Bowtie 2 paired-end alignment for CTread1GAread2CTgenome (reading in sequences from 95M_R1.fastp-trim.fq.gz_C_to_T.fastq and 95M_R2.fastp-trim.fq.gz_G_to_A.fastq, with the options: -q --score-min L,0,-1.0 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --norc)) Found first alignment: LH00469:254:22HGFVLT4:2:1101:1623:1014_1:N:0:TGAAGACG+ACATGCCA/1 77 * 0 0 * * 0 0 TATGAGGTGATGGTAAGAATAATATTGTTAGATAGATATGTAAGATTTATAATTATTGTATAAATTTAATGTATTTGATATATTGTTTAAAGTATTTTATAAATATGTAATAATGAAAAAGTAATGTAAAG IIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIII9IIIIIII9II-IIIIIIIIIIIIIIIIIIIII YT:Z:UP LH00469:254:22HGFVLT4:2:1101:1623:1014_2:N:0:TGAAGACG+ACATGCCA/2 141 * 0 0 * * 0 0 TAATAACTTAAATATTATCTCATTTATATCTCATACCTTATCTTCTTATACATTATATCTATCTAACCATAACCTTATTAAACAAATCTTTACATTACTTTTTCATTATTAAATATTTATAAAATACTTTA IIIIII9IIII9IIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIII YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 95M_R1.fastp-trim.fq.gz_C_to_T.fastq and 95M_R2.fastp-trim.fq.gz_G_to_A.fastq, with the options: -q --score-min L,0,-1.0 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --nofw)) Found first alignment: LH00469:254:22HGFVLT4:2:1101:1623:1014_1:N:0:TGAAGACG+ACATGCCA/1 77 * 0 0 * * 0 0 TATGAGGTGATGGTAAGAATAATATTGTTAGATAGATATGTAAGATTTATAATTATTGTATAAATTTAATGTATTTGATATATTGTTTAAAGTATTTTATAAATATGTAATAATGAAAAAGTAATGTAAAG IIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIII9IIIIIII9II-IIIIIIIIIIIIIIIIIIIII YT:Z:UP LH00469:254:22HGFVLT4:2:1101:1623:1014_2:N:0:TGAAGACG+ACATGCCA/2 141 * 0 0 * * 0 0 TAATAACTTAAATATTATCTCATTTATATCTCATACCTTATCTTCTTATACATTATATCTATCTAACCATAACCTTATTAAACAAATCTTTACATTACTTTTTCATTATTAAATATTTATAAAATACTTTA IIIIII9IIII9IIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIII YT:Z:UP >>> Writing bisulfite mapping results to 95M_pe.bam <<< Reading in the sequence files ../data/95M_R1.fastp-trim.fq.gz and ../data/95M_R2.fastp-trim.fq.gz Processed 1000000 sequence pairs so far Processed 2000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1120:39948:23118_1:N:0:TGAAGACG+ACATGCCA NW_026963585.1 2 Processed 3000000 sequence pairs so far Processed 4000000 sequence pairs so far Processed 5000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1146:28813:7289_1:N:0:TGAAGACG+ACATGCCA NW_026963415.1 1 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1150:21303:24911_1:N:0:TGAAGAAG+ACATGCCA NW_026963365.1 1 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1150:8955:27125_1:N:0:TGAAGACG+ACATGCCA NW_026963611.1 2 Processed 6000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1152:43096:15008_1:N:0:TGAAGAAG+ACATGCCA NW_026963743.1 1 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1155:47506:14882_1:N:0:TGAAGACG+ACATGCCA NW_026963415.1 3 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1156:5823:9545_1:N:0:TGAAGACG+ACATGCCA NW_026963738.1 23712 Processed 7000000 sequence pairs so far Processed 8000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1176:3331:24169_1:N:0:TGAAGACG+ACATGCCA NW_026963795.1 2 Processed 9000000 sequence pairs so far Processed 10000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1194:2667:24029_1:N:0:TGAAGAAG+ACATGCCA NW_026963596.1 19315 Processed 11000000 sequence pairs so far Processed 12000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1211:10322:1953_1:N:0:TGAAGACG+ACATGCCA NW_026963307.1 2 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1211:2028:16647_1:N:0:TGAAGACG+ACATGCCA NW_026963307.1 2 Processed 13000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1219:12022:17109_1:N:0:TGAAGACG+ACATGCCA NW_026963585.1 49251 Processed 14000000 sequence pairs so far Processed 15000000 sequence pairs so far Processed 16000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1247:18682:28021_1:N:0:TGAAGACG+ACATGCCA NW_026963688.1 1 Processed 17000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1249:19337:5202_1:N:0:TGAAGACG+ACATGCCA NW_026963725.1 1 Processed 18000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1260:14708:11758_1:N:0:TGAAGACG+ACATGCCA NW_026963679.1 1 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1260:18075:16353_1:N:0:TGAAGACG+ACATGCCA NW_026963415.1 2 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1260:18091:16353_1:N:0:TGAAGACG+ACATGCCA NW_026963415.1 2 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1264:11196:13271_1:N:0:TGAAGACG+ACATGCCA NW_026963562.1 2 Processed 19000000 sequence pairs so far Processed 20000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1278:22250:12654_1:N:0:TGAAGAAG+ACATGCCA NW_026963684.1 18481 Processed 21000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1287:40539:11254_1:N:0:CGAAGACG+ACATGCCA NW_026963693.1 21920 Processed 22000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1303:44447:21185_1:N:0:TGAAGACG+ACATGCCA NW_026963325.1 2 Processed 23000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1309:35505:4194_1:N:0:TGAAGACG+ACATGCCA NW_026963808.1 2 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1312:43654:23847_1:N:0:TGAAGACG+ACATGCCA NW_026963749.1 2 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1312:43662:23861_1:N:0:TGAAGACG+ACATGCCA NW_026963749.1 2 Processed 24000000 sequence pairs so far Processed 25000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1326:38329:19028_1:N:0:TGAAGACG+ACATGCCA NW_026963645.1 2 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1326:38305:19042_1:N:0:TGAAGAAG+ACATGCCA NW_026963645.1 2 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1327:41024:29072_1:N:0:TGAAGACG+ACATGCCA NW_026963390.1 1 Processed 26000000 sequence pairs so far Processed 27000000 sequence pairs so far Processed 28000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1353:48946:1462_1:N:0:TGAAGACG+ACATGCCA NW_026963585.1 2 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1355:9562:21031_1:N:0:TGAAGACG+ACATGCCA NW_026963480.1 1 Processed 29000000 sequence pairs so far Processed 30000000 sequence pairs so far Processed 31000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1380:31071:18061_1:N:0:TGAAGACG+ACATGCCA NW_026963443.1 56341 Processed 32000000 sequence pairs so far Processed 33000000 sequence pairs so far Processed 34000000 sequence pairs so far Processed 35000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1417:7110:12444_1:N:0:TGAAGACG+ACATGCCA NW_026963415.1 2 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1420:39373:10161_1:N:0:TGAAGACG+ACATGCCA NW_026963415.1 2 Processed 36000000 sequence pairs so far Processed 37000000 sequence pairs so far Processed 38000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1438:8833:26998_1:N:0:TGAAGACG+ACATGCCA NW_026963575.1 32005 Processed 39000000 sequence pairs so far Processed 40000000 sequence pairs so far Processed 41000000 sequence pairs so far Processed 42000000 sequence pairs so far Processed 43000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1487:27000:1882_1:N:0:TGAAGACG+ACGTGCCA NW_026963749.1 2 Processed 44000000 sequence pairs so far Processed 45000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2106:46802:10385_1:N:0:TGAAGACG+ACATGCCA NW_026963743.1 1 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2109:48898:10007_1:N:0:TGAAGACG+ACATGCCA NW_026963443.1 1 Processed 46000000 sequence pairs so far Processed 47000000 sequence pairs so far Processed 48000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2134:10274:12851_1:N:0:TGAAGACG+ACATGCCA NW_026963307.1 1 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2137:5475:11380_1:N:0:TGAAGACG+ACATGCCA NW_026963585.1 2 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2138:43565:2793_1:N:0:TGAAGACG+ACATGCCA NW_026963362.1 1 Processed 49000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2147:11617:6575_1:N:0:TGAAGACG+ACATGCCA NW_026963322.1 898872 Processed 50000000 sequence pairs so far Processed 51000000 sequence pairs so far Processed 52000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2169:40587:17165_1:N:0:TGAAGACG+ACATGCCA NW_026963585.1 2 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2171:16950:17571_1:N:0:TGAAGAAG+ACATGCCA NW_026963684.1 18476 Processed 53000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2177:26353:1490_1:N:0:TGAAGACG+ACATGCCA NW_026963305.1 1 Processed 54000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2186:27615:17683_1:N:0:TGAAGAAG+ACATGCCA NW_026963684.1 18469 Processed 55000000 sequence pairs so far Processed 56000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2212:12337:21858_1:N:0:TGAAGACG+ACATGCCA NW_026963456.1 1 Processed 57000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2226:24969:23833_1:N:0:TGAAGACG+ACATGCCA NW_026963808.1 2 Processed 58000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2228:47959:22782_1:N:0:TGAAGACG+ACATGCCA NW_026963307.1 124882 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2228:47967:22796_1:N:0:TGAAGACG+ACATGCCA NW_026963307.1 124882 Processed 59000000 sequence pairs so far Processed 60000000 sequence pairs so far Processed 61000000 sequence pairs so far Processed 62000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2273:2262:16521_1:N:0:TGAAGACG+ACATGCCA NW_026963585.1 49256 Processed 63000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2282:28344:19364_1:N:0:TGAAGACG+ACATGCCA NW_026963598.1 2 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2282:28360:19364_1:N:0:TGAAGACG+ACATGCCA NW_026963598.1 2 Processed 64000000 sequence pairs so far Processed 65000000 sequence pairs so far Processed 66000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2307:26919:4852_1:N:0:TGAAGACG+ACATGCCA NW_026963415.1 1 Processed 67000000 sequence pairs so far Processed 68000000 sequence pairs so far Processed 69000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2334:43573:6309_1:N:0:TGAAGACG+ACATGCCA NW_026963443.1 56338 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2337:41550:14433_1:N:0:TGAAGACG+ACATGCCA NW_026963415.1 2 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2338:23351:28904_1:N:0:TGAAGACG+ACATGCCA NW_026963377.1 2 Processed 70000000 sequence pairs so far Processed 71000000 sequence pairs so far Processed 72000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2368:23424:21577_1:N:0:TGAAGACG+ACATGCAA NW_026963340.1 4220809 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2370:22946:6659_1:N:0:TGAAGACG+ACATGCCA NW_026963684.1 18467 Processed 73000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2375:5702:25808_1:N:0:TGAAGACG+ACATGCCA NW_026963585.1 1 Processed 74000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2383:43266:19560_1:N:0:TGAAGACG+ACATGCCA NW_026963415.1 2 Processed 75000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2403:41396:26690_1:N:0:TGAAGACG+ACATGCCA NW_026963298.1 2989484 Processed 76000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2413:44318:13817_1:N:0:TGAAGACG+ACATGCCA NW_026963308.1 1 Processed 77000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2426:27907:18888_1:N:0:TGAAGACG+ACATGCCA NW_026963590.1 1 Processed 78000000 sequence pairs so far Processed 79000000 sequence pairs so far Processed 80000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2453:21481:8382_1:N:0:TGAAGACG+ACATGCCA NW_026963415.1 2 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2453:21473:8396_1:N:0:TGAAGACG+ACATGCCA NW_026963415.1 2 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2453:21490:8396_1:N:0:TGAAGACG+ACATGCCA NW_026963415.1 2 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2456:6665:7388_1:N:0:TGAAGACG+ACATGCCA NW_026963415.1 1 Processed 81000000 sequence pairs so far Processed 82000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2475:26491:23665_1:N:0:TGAAGAAG+ACATGCCA NW_026963307.1 124921 Processed 83000000 sequence pairs so far Processed 84000000 sequence pairs so far Processed 85000000 sequence pairs so far 85285639 reads; of these: 85285639 (100.00%) were paired; of these: 36422528 (42.71%) aligned concordantly 0 times 16474693 (19.32%) aligned concordantly exactly 1 time 32388418 (37.98%) aligned concordantly >1 times 57.29% overall alignment rate 85285639 reads; of these: 85285639 (100.00%) were paired; of these: 36431256 (42.72%) aligned concordantly 0 times 16464083 (19.30%) aligned concordantly exactly 1 time 32390300 (37.98%) aligned concordantly >1 times 57.28% overall alignment rate Processed 85285639 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 341142556. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 341142556. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 341142556. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 341142556. Successfully deleted the temporary files 95M_R1.fastp-trim.fq.gz_C_to_T.fastq and 95M_R2.fastp-trim.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 85285639 Final Cytosine Methylation Report ================================= Total number of C's analysed: 2062598827 Total methylated C's in CpG context: 28559323 Total methylated C's in CHG context: 3156055 Total methylated C's in CHH context: 14721005 Total methylated C's in Unknown context: 372421 Total unmethylated C's in CpG context: 212524586 Total unmethylated C's in CHG context: 328121055 Total unmethylated C's in CHH context: 1475516803 Total unmethylated C's in Unknown context: 11532776 C methylated in CpG context: 11.8% C methylated in CHG context: 1.0% C methylated in CHH context: 1.0% C methylated in unknown context (CN or CHN): 3.1% Bismark completed in 0d 6h 1m 0s ==================== Bismark run complete ==================== Unable to flush stdout: Broken pipe